Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/644—Coagulation factor IXa (3.4.21.22)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21022—Coagulation factor IXa (3.4.21.22)

Definitions

• This invention is in the field of genetic engineering relating to factor IX DNA. 2. Description of prior art
• Factor IX Greek factor or antihaemophilic factor B
• zymogen of a serine protease which is required for blood coagulation via the intrinsic pathway of clotting (Jackson & Nemerson, Ann.Rev.Biochem. 49 , 765-811, 1980). This factor is synthesised in the liver and requires vitamin K for its biosynthesis (Di Scipio & Davie, Biochem. 18, 899-904, 1979).
• Human factor IX has been purified and characterised, but details of the amino acid sequence are fragmentary. It is a single-chain glycoprotein, with a molecular weight of approximately 60,000 (Suomela, Eur.J.Biochem. 71, 145-154, 1976). Like other vitamin K-dependent plasma proteins, human factor IX contains in the amino-terminal region approximately 12 gamma-carboxyglutamic acid residues (Di Scipio & Davie, Biochem. 18, 899-904, 1979).
• factor IX is acted upon by activated factor XI (XIa) by the cleavage of two internal peptide bonds, releasing an activation glycopeptide of 10,000 daltons (Di Scipio et al. , J.Clin. Invest. 61. 1528-1538, 1978).
• the activated factor IX (IXa) is composed of two chains held together by at least one disulphide bond.
• Factor IXa then participates in the next step in the coagulation cascade by acting on factor X in the presence of activated factor VIII, Ca ++ ions, and phospholipids (Lindquist et__al;, J.Biol.Chem. 253, 1902-1909, 1978).
• the current method of diagnosing Christmas disease involves measurement of the titre of factor IX in plasma by a combination of a clotting assay and an immunochemical assay.
• Treatment of haemorrhage in the disease consists of factor IX replacement by means of intravenous transfusion of human plasma protein concentrates enriched in factor IX.
• the enrichment of plasma in factor IX is a time-consuming process. Summary of the invention
• the invention arises from the finding that an extensive DNA sequence of the human factor IX genome can be obtained by a clever and laborious combination of chemical synthesis and artificial biosynthesis, starting from elementary nucleotide or dinucleotide "building blocks", as will be described below.
• a major feature of the invention comprises recombinant DNA which comprises a cloning vehicle DNA sequence and a sequence foreign thereto (i.e. foreign to the vehicle) which is substantially the same as a sequence occurring in the human factor IX genome.
• a sequence foreign thereto i.e. foreign to the vehicle
• a 11873 nucleotide long part of such a foreign sequence has been identified and a very large part of It has been sequenced by the Maxam-Gilbert sequencing method.
• a 129 nucleotide length of this sequence is more than sufficient to characterise it unambiguously as coding for a specific protein and a particular such length is regarded herein as useful to characterise the whole sequence inserted in the cloning vehicle as one occurring in the human factor IX genome.
• a further feature of the Invention therefore comprises recombinant DNA which comprises a cloning vehicle or vector DNA sequence and a DNA sequence foreign thereto which consists of or includes substantially the following sequence of 129 nucleotides (which should be read In rows of 30 across the page):-
• the invention includes particularly recombinant DNA which comprises a cloning vehicle DNA sequence and a sequence foreign to the cloning vehicle, wherein the foreign sequence includes substantially the whole of an exon sequence of the human factor IX genome.
• the 129-nucleotide sequence described above corresponds substantially to such an exon sequence.
• Another such exon sequence which independently characterises the human factor IX DNA is the 203-nucleotide sequence substantially as follows (again reading In rows of 30 across the page):-
• the intron sequences of the human factor IX genome are excised during the transcription process by which mRNA is made in human cells. Only exon sequences are translated into protein.
• DNA coding for factor IX has been prepared from human mRNA. This cDNA has been partly sequenced and found to contain the same 129- and 203-nucleo tide sequences set out above.
• the invention also includes recombinant DNA which comprises a cloning vehicle sequence and a DNA sequence foreign to the cloning vehicle, wherein the foreign sequence comprises a DNA sequence which is complementary to human factor IX mRNA.
• Such a recombinant cDNA can be isolated from a library of recombinant cDNA clones derived from human liver mRNA by using an exon of the genomic human factor IX DNA (or part thereof) as a probe to screen this library and thence isolating the resulting clones.
• the invention also includes recombinant DNA in which the foreign sequence is any fragment of human factor IX DNA, particularly of length at least 50 and preferably at least 75 nucleotides or base-pairs. It includes such recombinant DNA whether or not part of the 129 or 203-base-pair sequence defined above. It includes especially part or all of the exon sequences of human factor IX genomic DNA. Various short lengths up to about 11 kilobases (11,000 nucleotides or base-pairs) long have been prepared by use of various restriction endonucleases. Methods of isolating recombinant DNA from clones are well known and some are described hereinafter.
• the DNA of the Invention can be single or double stranded form.
• the recombinant human factor IX DNA of this invention is useful as a tool of recombinant DNA technology. Thus it is useful as the first stage in the production of artificial human factor IX and in the preparation of probes for diagnostic purposes.
• appropriate cDNA or genomic clones will be introduced into a suitable expression vector in either mammalian or bacterial systems.
• the gene might be too long to be conveniently retained in one clone. Therefore a suitable artificial "minigene" will be designed and constructed from suitable parts of the cDNA and genomic clones.
• the minigene will be under the control of its own promoter or instead will be replaced by an artificial one, perhaps the mouse metallothioneine I promoter.
• the resultant 'minigene' will then be introduced into mammalian tissue culture cells e.g. a hepatoma cell line, and selection for clones of cells synthesising maximum amounts of biologically active factor IX will be carried out.
• mammalian tissue culture cells e.g. a hepatoma cell line
• selection for clones of cells synthesising maximum amounts of biologically active factor IX will be carried out.
• "genetic farming” could be employed as has been demonstrated for mouse growth hormone (Palmiter et al, Nature 300, 611-615, 1982).
• the minigene would be micro-injected into the pronucleus of fertilised eggs, followed by in vivo cloning and selection for progeny producing the largest quantity of human factor IX in blood.
• the cDNA clone or selected parts of it will be linked to a suitable strong bacterial promotor, e.g. a Lac or Trp promotor or the lamdba P R or P L ,
• the natural factor IX polypeptide is synthesised as a precursor containing both a signal and propeptide region. They are both normally cleaved off In the production of the definitive length protein. Even this product is merely a precursor. It is biologically inactive and must be gamma-carboxylated at 12 specific N-terminal glutamic acid residues in the so called 'GLA' domain by the action of a specific vitamin K-dependent carboxylase. In addition, two carbohydrate molecules are added to the connecting peptide region of the molecule, but it remains unknown whether they are required for activity. The substrate for the carboxylase is unknown and could be the precursor factor IX polypeptide or alternatively the definitive length protein.
• polypeptides both with and without the precursor domains will be "constructed” using genetic engineering methods in bacterial hosts. They will then be tested as substrates for the conversion of inactive to biologically active factor IV in vitro by the action of partially purified preparations of the carboxylase enzyme which can be isolated from liver microsomes or other suitable sources.
• the recombinant human genomic factor IX DNA or recombinant human mRNA-derived factor IX DNA has a wide variety of uses. It can be cleaved by enzymes or combinations of two or more enzymes into shorter fragments of DNA which can be recombined into the cloning vehicle, producing "sub-clones".
• probe DNA (by definition) is labelled in some way, conveniently radiolabelled, and can be used to examine in detail mutations in the human DNA which ordinarily would produce factor IX.
• probes have been produced for examining several different regions of the genome where mutation was suspected to have occurred in patients. Failure to obtain hybridisation from such a probe indicates that the sequence of the probe differs in the patient's DNA. In particular it has been shown that Christmas disease can be detected or confirmed by such methodology.
• Useful probes can contain intron and/or exon regions of the genomic DNA or can contain cDNA derived from the mRNA.
• the invention includes particularly probe DNA, i.e. which is labelled, and of a length suitable for the probing use envisaged. It can be single-stranded or double-stranded over at least the human factor IX DNA probing sequences thereof and such sequences will usually have a length of at least 15 nucleotides, preferably at least 19-30 nucleotides ⁇ n order to have a reasonable probability of being unique They will not usually be larger than 5 kb and rarely longer than 10 kb.
• the Invention accordingly includes a DNA molecule, comprising part of the human factor IX DNA sequence, whether or not labelled, whether intron or exon or partly both. It also Includes human cDNA corresponding to part of all of human factor IX mRNA. It includes particularly a solution of any DNA of the invention, which is a form in which it is conveniently obtainable by electroelution from a gel.
• the invention includes, of course, a host transformed with any of the recombinant DNA of the invention.
• the host can be a bacterium, for example an appropriate strain of E.coli, chosen according to the nature of the cloning vehicle employed.
• Useful hosts may include strains of Pseudomonas, Bacillus subtilis and
• Bacillus stearothermophilus Bacillus stearothermophilus, other Bacilli, yeasts and other fungi and mammalian (Including human) cells.
• One process practised in connection with this invention for preparing a host transformed with the recombinant DNA of the invention is based on the following steps:- (1) synthesising an oligodeoxynucleotide having a nucleotide sequence comprising that occurring in bovine factor IX messenger RNA coding for amino acids 70-75 or 348-352 of bovine factor IX, and labelling the oligodeoxynucleotide to form a probe; (2) preparing complementary DNA to a mixture of bovine mRNAs;
• step 2 (5) probing the clones with the synthetic oligodeoxynucleotide probe obtained in step 1 and isolating the resultant recombinant bovine factor IX cDNA-containing clone; (6) digesting the recombinant bovine factor IX cDNA from said clone with one or more enzymes to produce a bovine factor IX cDNA molecule comprising a shorter sequence of bovine factor IX DNA, but preferably at least 50 base-pairs long; and
• Figure 1 shows the structure of a published amino-acid sequence of bovine factor IX polypeptide, the deduced sequence of the mRNA from which it would be translated and the structures of oligonucleotides (oligo-N1 and N2) synthesised in the course of this invention
• Figures 2 and 3 show the chemical formulae of "building blocks" used to synthesise the oligonucleotides referred to in Figures 1 and 11;
• Figure 4 is an elevational view, partly sectioned, showing an apparatus for synthesising oligonucleotides
• Figure 5 shows the sequence of part of the bovine factor IX cDNA obtained in this invention
• Figure 6 is a map showing the organisation of an approximately 27 kb length of human factor IX genomic DNA and is divided into five portions, showing: - (a) the exon regions; (b) the 11,873- nucleotide length sequenced;
• Figure 7 shows the sequence of the DNA of Figure 6(b) and in parts the encoded protein;
• Figure 8 shows a restriction enzyme chart of the sequence shown in Figure 7;
• Figure 9 shows part of the sequence of the human factor IX cDNA and its encoded protein
• Figure 10 shows the structure of a pair of complementary oligonucleotides (oligo N3 and N4) synthesised in the course of this invention
• Figure 11 shows part of the DNA sequence of the vector pAT153/PvuII/8 of this invention, in the region where it differs from pAT153;
• Figure 12 is a diagram of plasmid pHIX17 of the invention showing the origin of the 1.4 kb fragment used for probing and initial sequencing; and
• Figure 13 shows the position of the major radioactive bands on probing a "Southern blot" of normal human DNA, cut by the restriction enzymes EcoRI(E), HindIII(H), BglII(B) and Bcll (Bc), with a sub-clone of the recombinant human factor IX DNA of this invention.
• a recombinant DNA of the Invention can be extracted by means of probes from a library of cloned human genomic DNA. This is a known recombinant library and the invention does not, of course, extend to human genomic factor IX DNA when present In such a library.
• the probes used were of bovine factor IX cDNA (DNA complementary to bovine mRNA) , which were prepared by an elaborate process involving firstly the preparation of recombinant bovine cDNA from a bovine mRNA starting material, secondly the chemical syntheses of oligonucleotides, thirdly their use to probe the recombinant bovine cDNA, in order to extract bovine factor IX cDNA and fourthly the preparation of suitable probes of shorter length from the recombinant bovine factor IX cDNA.
• the first probe tried appeared to contain an irrelevant sequence and the second probe tried, not containing it, proved successful in enabling a single clone of the human genomic factor IX DNA to be Isolated.
• This clone is designated lambda HIX-1.
• the steps involved are described in more detail in the sub-section "Examples" appearing hereinafter, and the second probe comprises the 247 base-pair DNA sequence of bovine factor IX cDNA indicated in Figure 5 of the drawings.
• the invention therefore provides specifically a recombinant DNA which comprises a cloning vehicle sequence and a DNA sequence foreign to the cloning vehicle, which recombinant DNA hybridises to a 247 base-pair sequence of bovine factor IX cDNA indicated in Figure 5 (by the arrows at each end thereof).
• the cloning vehicle or vector employed in the invention can be any of those known in the genetic engineering art (but will be chosen to be compatible with the host). They include E.coli. plasmids, e.g. pBR322, pAT153 and modifications thereof, plasmids with wider host ranges, e.g. RP4 plasmids specific to other bacterial hosts, phages, especially lambda phage, and cosmids.
• a cosmid cloning vehicle contains a fragment of phage DNA Including its cos (cohesive-end site) inserted in a plasmid. The resultant recombinant DNA is circular and has the capacity to accommodate very large fragments of additional foreign DNA.
• Fragments of human factor IX genomic DNA can be prepared by digesting the cloned DNA with various restriction enzymes. If desired, the fragments can be religated to a cloning vehicle to prepare further recombinant DNA and thereby obtain "sub-clones".
• a new cloning vehicle has been prepared.
• This is a modified pAT153 plasmid prepared by ligating a BamHI and HindIII double digest of pAT153 to a pair of complementary double sticky-ended oligonucleotides having a DNA sequence providing a BamHI restriction residue at one end, a HindIII restriction residue at the other end and a PvuII restriction site in between.
• the invention includes human factor IX cDNA (complementary to human factor IX mRNA) which contains substantially the same sequences.
• a library of human cDNA has been prepared and probed with human factor IX genomic DNA to isolate human factor IX cDNA from the library.
• the probe DNA is conveniently of relatively short length and must include at least one exon sequence.
• E.coli K-12 strain HB 101 Boyer & Roulland-Dussoix, J.Mol. Biol. 41, 459-472, 1969
• E.coli K-12 strain K803 which is a known strain used by genetic engineers.
• the mRNA preparation was passaged through an oligo dT-cellulose column (Caton and Robertson, Nucl.Acids Res. 7, 1445-1456, 1979) to isolate poly(A) + mRNA.
• Poly(A) + mRNA was translated in a rabbit reticulocyte cell-free system in the presence of 35 S-cysteine as described by Pelham and
• factor IX polypeptide was precipitated by the addition of specific anti-factor IX immunoglobulins.
• the immunoprecipitation procedure was as described by Choo et al. , Biochem.J. 181, 285-294, 1979.
• the immunoprecipitated material was washed throughly and resolved on a two-dimensional SDS-poly-acrylaraide gel (Choo et al., Biochem.J. 181, 285-294, 1979), by isoelectric focussing in one dimension and electrophoresis in another.
• Some polypeptides of known molecular weight were subjected to this procedure, to serve as reference points.
• the immunoprecipitated material showed 4 pronounced spots, all in the 50,000 molecular weight region and with separated isoelectric points. These predominant spots of molecular weight about 50,000 represent a single polypeptide chain plus a possible prepeptide signal sequence, a deduction compatible with published data (Katayama et al.,Proc. Natl.Acad. Sci.USA 76. 4990-4994, 1979).
• oligo N2A and oligo N2B were those known to differ in sequence in the analogous serine proteases, prothrombin, Factor C and Factors VII and X and were those corresponding to amino acids 70-75 and 348-352 respectively.
• the 70-75 region was particularly favourable in that the mixture of oligonucleotides synthesised, i.e. oligo N2A and oligo N2B, contained all 16 possible sequences that might occur in a 17 nucleotide long region of the mRNA corresponding to amino acids 70-75.
• the oligo N2A-N2B mixture is hereinafter called "oligo N2" for brevity.
• Figure 1 of the drawings shows the two selected regions of the known amino acid sequence of bovine factor IX, the corresponding mRNA and the oligonucleotides synthesised. Since some of the amino acids are coded for by more than one nucleotide triplet, there are 4 ambiguities in the mRNA sequence shown for amino acids 70-75 and therefore 16 possible individual sequences.
• nucleotide mixtures oligo N1 and oligo N2 were synthesized using the solid phase phosphotriester method of Duckworthet al. , Nucl.Acids Res. 9, 1691-1706, 1981, modified in two ways. Firstly, o-chlorophenyl rather than p-chlorophenyl blocking groups were used for the phosphotriester grouping, and were Incorporated in the mononucleotide and dinucleotide "building blocks".
• Figures 2 and 3 of the drawings show (a) dinucleotide and (b) mononucleotide "building blocks”.
• DMT 4,4' - dimethoxytrityl
• B 6-N-benzoyladenin-9-yl, 4-N-benzoylcytosin-1-yl, 2-N-isobutyrylguanin-9-yl or thymin-1-yl, depending on the nucleotide selected.
• reaction cell used for the successive addition of mono- or dinucleotide "building blocks” was miniaturised so that the coupling step with the condensing agent 1-(mesitylene-2-sulphonyl)-3-nitro-1,2,4-triazole (MSNT) was carried out in a volume of 0.5ml pyridine containing 3.5 micromoles of polydimethylacrylamide resin, 17.5 micromoles of incoming dinucleotide (or 35 micromoles of mononucleotide) and 210 micromoles of MSNT.
• MSNT condensing agent 1-(mesitylene-2-sulphonyl)-3-nitro-1,2,4-triazole
• Figure 4 of the drawings is an elevational view of the micro-reaction cell 1 and stopper 2 used for oligonucleotide synthesis, drawn 70% of actual size.
• the device comprises a glass-to-PTFE tubing joint 3 at the inlet end of the stopper 2.
• the stopper has an internal conduit 4 which at its lower end passes into a hollow tapered ground glass male member 5 and thence into a sintered glass outlet 6 to the stopper.
• the cell 1 has a ground glass female member 7 complementary to the member 5 of the stopper, leading to reaction chamber 8, the lower end of which terminates in a sintered glass outlet 9. This communicates with glass tubing 10, and a 1.2mm. "Interflow" tap 11. Further glass tubing 10, beyond the tap 11, leads to the outlet glass-to-PTFE tubing joint 12.
• Pairs of ears 13 on the stopper and cell enable them to be joined together by springs (not shown) in a liquid-tight manner.
• fractiona tion was carried out by high pressure liquid chromatography (Duckworth et_ al. , see above) and the peak tubes corresponding to the product of correct chain length were located by labelling of fractions at their 5'-hydroxyl ends using [gamma- 32 P]-ATP and T4 polynucleotide kinase, followed by 20% 7M urea polyacrylamide gel electrophoresis.
• dNTP denotes the mixture of 4 deoxynucleoside triphosphates required for synthesis. Oligo N-1 hybridises to the corresponding region on the mRNA (refer to Figure 1) and thereby acts as a primer for the initiation of transcription. It was used in order to achieve a further enrichment for factor IX mRNA.
• the cDNA was extracted with phenol and desalted on a Sephadex-G100 column, before It was treated with alkali (0.1M NaOH, ImM EDTA) for 30 min. at 60°C to remove the mRNA strand. Second strand DNA synthesis was then carried out exactly as published (Huddleston & Brownlee, Nucl.Acids Res. 10, 1029-1038, 1981).
• the double-stranded DNA was next cleaved with the restriction enzyme Mbol and ligated to the plasmid vector pBR322 which had been cut with BamHI and treated with calf intestinal alkaline phosphatase to minimise vector self-religation.
• Phosphatase treatment was carried out by incubating 5 micrograms of BamHI-cut pBR 322 plasmid with 0.5 microgram calf intestinal phosphatase (Boehringer; in 10mM Tris - HCl buffer, pH 8.0) in a volume of 50 microlitres at 37oC for 10 minutes, see Huddleston & Brownlee supra.
• E.coli strain MC 1061 The ligated DNA was used to transform E.coli strain MC 1061.
• E.coli MC 1061 was grown to early exponential phase as Indicated by an absorbancy of 0.2 at 600 nm and made "competent" by treating the pelleted bacterial cells first with one half volume, followed by repelleting, and then with 1/50 volume of the original growth medium of 100mM CaCl 2 15% v/v glycerol and 10mM PIPES-NaOH, pH 6.6 at 0°C. Cells were immediately frozen in a dry ice/ethanol bath to -70°C.
• dC/dG tailed library In the preparation of this library, first strand cDNA was synthesised as described for the above library except that oligo dT ( 12-18) was used as a primer to initiate cDNA synthesis. Following this, the cDNA was tailed with dCTP using terminal transferase and back-copied with the aid of oligo dG (12-18) primer and reverse transcriptase to give double stranded DNA, exactly according to the method of Land et al. , Nucl.Acids Res. 9 , 2251-2266, 1981.
• the library of colonies In an unordered fashion, was transferred onto 13 Whatman 541 filter papers and amplified with chloramphenicol, to Increase the number of copies of the plasmid in the colonies, as described by Gergen et al., Nucl. Acids Res., 1, 2115-2136 (1979).
• Hybridisation was carried out at 47oC for 20h in the same solution containing 3 x 10 5 cpm (0.7 nanogram/ml) of labelled oligo N-2 probe. Labelling was done by phosphorylation of the oligonucleotides at the 5' hydroxyl end using [gamma- 32 P] -ATP and T4 phosphokinase (Huddleston & Brownlee,
• brackets in Figure 5 does not agree with the published bovine factor IX amino acid data of Katayama.
• the underlined portion denotes the sequence corresponding to the oligo N-2 probe sequence
• the asterisk denotes a nonsense codon
• the brackets enclose a sequence which does not correspond to Katayama's amino acid data
• the arrows indicate Hinfl restriction sites.
• the Katayama numbering system for amino acids is shown and this sequence is in the opposite orientation to the direction of transcription of the tetracycline-resistant gene of the plasmid.
• a library of cloned human genomic DNA namely a Haelll/Alul lambda phage Charon 4A library prepared by Lawn et al. , Cell, 15, 1157-1174, 1978, was used. 10 phage recombinants from this library were screened using the in situ plaque hybridisation procedure as described by T. Maniatis et a l . , Cell, 15, 687, 1978. Pre-hybridisation and hybridisation were carried out at 42oC In 50% formamide.
• the first fragment corresponds to nucleotide numbers -8 to 317 on the numbering system of Figure 5, and was isolated by Sau3AI digestion of BIX-1 plasmid DNA.
• the isolated DNA was labelled to high specific activity by incorporation of [alpha - 32 P] -dATP using a nick translation (Rigby et al.,
• the initial lambda HIX-1 clone was characterised by cleavage with various single and double digests with different restriction endonucleases and Southern blotting of fragments using the bovine factor IX cDNA probe (results omitted for brevity).
• the subsequently Isolated lambda HIX-2 and 3 clones were characterised in the same way except that the human cDNA probe, pATIXcVII (see Section L below) was used for the Southern blots. From these results it emerged that the sequences in the factor IX genome corresponding to lambda HIX-2 and 3 overlapped with lambda HIX-1 as shown in Figure 6(e).
• Section (d) of this Figure 6 are summarised the results of the analysis using the restriction enzymes EcoRI (E) , HindiII (H) , Bglll (B) , BamHI (Ba) and PvuII (P) , and this serves as a restriction enzyme map.
• EcoRI EcoRI
• H HindiII
• Bglll Bglll
• BamHI Ba
• PvuII P
• Sequencing Numerous sub-clones were isolated from a knowledge of the rectriction enzyme map as described in Section J(ii) below, the majority in a vector pAT153/PvuII/8.
• Figure 7 shows the sequence of one strand of the DNA.
• the nucleotides are arbitrarily numbered from 1 to 11873 in the 5' to 3' direction.
• the original 403-nucleotide sequence runs from Figure 7 nucleotides Nos. 4372 to 4774 and is indicated by 0-0'.
• the 129-nucleotide sequence lying within the 403 one runs from Figure 7 nucleotides Nos. 4442 to 4570 and is indicated by J-J'. This corresponds exactly to the "w" exon.
• Figure 8 is a chart produced by a computer analysis of the sequence data of the 11873 nucleotide long sequence of Figure 7.
• Column 1 of Figure 8 gives the arbitrary nucleotide number allotted to the nucleotide of Figure 7.
• Column 2 apportions the nucleotide number as a fraction of the whole sequence.
• Column 3 shows the restriction enzymes which will cut the DNA within various short sequences of nucleotides shown in Column 4.
• the short sequences of Column 4 begin with the nucleotide numbered in Column 1.
• sequences II-IV are produced by restriction at the following sites (denoted by the first nucleotide number at the 5' end of each site).
• Figure 8 Particularly important sites are arrowed in Figure 8.
• Figure 6(c) Some of the relevant nucleotide numbers are shown in Figure 6(c), the number given being that of the nucleotide at the 5' end of each site.
• Further sequence analysis of the sub-clones V, VI, VII and VIII shown in Figure 6(c) indicates that the factor IX gene is divided into at least 7 exon regions separated by at least 6 introns.
• the positions of the exons are shown In Figure 6(a) by the solid blocks labelled t, u, v, w, x, y and z .
• the "z" exon is much the longest and its 3'-end coincides with the 3'-end of the MRNA.
• pHIX-17 DNA was digested with EcoRI.
• the digested material was resolved on 0.8% agarose gel and a 1.4 kb fragment was isolated in solution by electroelution. It can be stored in the usual manner. This 1.4 kb long molecule was used for the initial sequencing. Only about 1.0 kb Is Inserted DNA, the remaining 0.4 kb being of pBR322.
• a 403 nucleotide length of the inserted DNA was sequenced and is Identified as 0—0' in
• oligo N3 Two partially complementary synthetic deoxyoligonucleotides, oligo N3, and, oligo N4, were synthesised by the solid phase phosphotriester method described in Section C above. Each has "overhanging" BamHI and Hindlll recognition sequences and an internal PvuII recognition sequence.
• Figure 10 shows the structures of oligo N3 and oligo N4. BamHI and HindIII cleave ds DNA to leave sticky or "overhanging" ends. For example Hindlll cleaves - AAGCTT - TTCGAA between the adenine-carrying nucleotides of each strand leaving the sticky-ended complementary strands :-
• pAT153 was digested with Hindlll and BamHI and the 3393 nucleotide long linear fragment was separated from the 346 nucleotide shorter fragment by 0.7% agarose gel electrophoresis, followed by electroelution of the appropriate bands visualised by ethidium bromide fluorescence under UV light.
• the plasmid DNA was then analysed for its ability to be linearised by the restriction enzymes BamHI, Hindlll and PvuII.
• Four clones were positive in this assay and one, labelled pAT153/PvuII/8, was selected for sequence analysis by the Maxam-Gilbert method across the newly constructed section of the plasmid. This part of the sequence is shown in Figure 11 along the unique restriction sites.
• the novel part of the plasmid sequence is underlined: the remainder is present in the parent plasmid pAT153.
• the vector allows blunt-end cloning (after treatment with phosphatase) into the inserted PvuII site.
• the cloned DNA can be excised, assuming that it lacks appropriate internal restriction sites, with BamHI/Hindlll,
• BamHI/Clal or BamHI/EcoRI double digests are also convenient for end labelling with 32 P for characterization of the ends of cloned DNA by the Maxam-Gilbert sequencing method.
• Plasmid DNA from pHIX-17 was prepared and cleaved with PvuII. Five discrete fragments, all derived from the DNA insert of pHIX-17, were isolated. The sizes of these fragments were approximately 2.3, 1.3, 1.2, 1.1 and 1.0 kilobases. These fragments were blunt-end ligated into the PvuII site of the pAT153/PvuII/8 vector and transformed into E. coli HB 101. Five clones of recombinant DNA which carried the 2.3, 1.3, 1.2, 1.1 and 1.0 kb fragments were obtained and these were designated pATIXPvu-1, 2, 3, 4 and 5 respectively.
• Factor IX DNA from pATIXPvu-2 is abbreviated as IV and pATIXPvu-5 as III in Figure 6(c).
• FIG. 6(d) pATIXEco-1 was further digested with both EcoRI and Bglll, and the "overhanging ends" of the restriction sites filled in with deoxynucleoside triphosphates using the Klenow fragment of DNA polymerase I. After isolation of the resulting 1.1 kb fragment by agarose gel electrophoresis and electroelution, it was blunt-end ligated using T4 DNA ligase into the PvuII site of pAT153/PvuII and allowed to transform E.coli MC 1061. The resultant sub-clone was designated pATIXBE and the factor IX DNA sequence thereof is abbreviated as II in Figure 6(c).
• the second strand synthesis was carried out by adding directly to the above solution 20 microlitres of 5x second strand buffer (250 mM Hepes/KOH pH 6.9, 250 mM KC1, 50 mM MgCl 2 ) , 4 microlitres of a 2.5 mM mixture of each of the four deoxynucleoside triphosphates, 10 microlitres of E.coli DNA polymerase I (6 units per microlitre) and making the volume of the solution up to 100 microlitres with deionized water.
• 5x second strand buffer 250 mM Hepes/KOH pH 6.9, 250 mM KC1, 50 mM MgCl 2
• 4 microlitres of a 2.5 mM mixture of each of the four deoxynucleoside triphosphates 10 microlitres of E.coli DNA polymerase I (6 units per microlitre) and making the volume of the solution up to 100 microlitres with deionized water.
• S 1 nuclease digestion was carried out by the addition of 400 microlitres of S 1 nuclease buffer (0.03 M sodium acetate pH 4.4, 0.25 M NaCl, 1 mM ZnSO 4 ) and 1 microlitre of S. nuclease (at 500 units per microlitre). After incubating for 30 minutes at 37oC, 10 microlitres of 0.5 M EDTA (pH 8.0) was added. Double stranded DNA was deproteinised by shaking with an equal volume of a phenol: chloroform (1:1) mixture, followed by ether extraction of the aqueous phase and precipitation of ds DNA by addition of 2 volumes of ethanol.
• DNA polymerase I "fill in" of S 1 ends was carried out by a further incubation of the sample dissolved In 25 microlitres of 50 mM tris-chloride, pH 7.5, 10 mM MgCl 2 , 5 mM dithiothreitol and containing 0.02 mM dNTP and 6 units of DNA polymerase I. After incubating for 10 minutes at room temperature, 5 microlitres of EDTA (0.1 M at pH 7.4) and 3 microlitres of 5% sodium dodecyl sulphate (SDS) were added.
• SDS sodium dodecyl sulphate
• the following part of the protocol differs from the 'Stanford protocol'.
• the sample was fractionated on a "mini"-Sephacryl S400 column run in a disposable 1 ml pipette In 0.2 M NaCl, 10 mM Tris-chloride, pH 7.5 and 1 mM EDTA.
• the first 70% of the "breakthrough" peak of radioactivity was pooled (0.4 ml) and deproteinised by shaking with an equal volume of n-butanol:chloroform (1:4).
• To the aqueous phase was added 1 microgram of yeast RNA (BDH) as carrier followed by 2 volumes of ethanol.
• yeast RNA yeast RNA
• This 20-22S cDNA library was amplified by growing the E.coli for a further 6 hours at 37°C. 1 ml aliquots of this amplified library were stored at -20oC in L broth containing 15% glycerol, before screening for factor IX cDNA clones.
• L. Isolation and sequence analysis of human factor IX cDNA clones 6000 colonies of the amplified 20-22S human cDNA library were plated on each of ten 15 cm agar plates and after growing overnight were blotted into Whatman 541 filter paper.
• nick-translation was carried out as before and the material used in a hybridisation reaction for 16 hours at 65oC in 3x SSC, 10x Denhardts solution, 0.1% SDS and 50 micrograms/ml sonicated denatured E.coli DNA and 100 micrograms/ml of sonicated denatured herring sperm DNA.
• hydridisation filters were washed at 65oC successively in 3x SSC, 0.1% SDS (2 changes, half an hour each) and 2x SSC, 0.1% SDS (2 changes, half an hour each).
• 7 clones were selected as positive, but on dilution followed by re-screening by hydridisation as above, only 5 proved to be positive.
• Nucleotides 1-130 were derived from clone pATIXcVI which extends from nucleotides 1-1548 of Figure 9.
• the sequence from Nos. 2002-2778 was derived by isolating 4 additional clones designated pATIX108.1, pATIX108.2, pATIX108.3 and pATIXDB.
• the first 3 were derived from a mini-library (designated GGB108) of cDNA clones constructed exactly as described in section K above except that sucrose density gradient centrifugation was used instead of chromatography on "Sephacryl" S-400 to fractionate the double-stranded DNA according to size. A fraction of m.w. from 1 kb-5 kb was selected and an amplified library of 10,000 independent clones containing approximately 20% background non-recombinant clones was obtained.
• Clone pATIXDB derived from another cDNA library (designated DB1) constructed as described in section K except that total poly A+ human liver mRNA was used as the starting material and sucrose density gradient centrifugation was used to fractionate the DNA according to size as in the construction of the mini-library GGB108.
• the complexity of this library was 95,000 with an estimated background of non-recomibinants of 50%.
• Clones pATIX108.1 and pATIXl08.2 were selected from a group of 30 hybridization-positive clones isolated by Grunstein-Hogness screening of the mini library GGB108 using a 32 P-nick translated probe derived from a Sau3AI restriction enzyme fragment, itself derived from nucleotides 1796-2002 of clone pATIXcVII. From pATIXl08.1 the sequence of nucleotides 2009-2756 was determined ( Figure 9). Following this the sequence of a part of pATIX108.2, specifically nucleotides 1950-2086, provided the overlap with pATIXcVII.
• clone pATIX108.3 was selected and sequenced from nucleotides 2642-2778. This clone was followed by three A nucleotides, which sequence was confirmed as a vestigial marker for the poly A tail, by the subsequent isolation of clone pATIXDB by a similar method.
• pATIXDB was sequenced from Nos. 2760-2778 and ended in 42 A nucleotides, thus marking the 3' end of the mRNA.
• Figure 9 shows that the predicted amino acid sequence codes for a protein of 456 amino acids, but included in this are 41 residues of precursor amino acid sequence preceding the N-terminal tyrosine residue of the definitive length factor IX protein.
• the precursor section of the protein shows a basic amino acid domain (amino acids -1 to -4) as well as the more usual hydrophobic signal peptide domain (amino acids -21 to -36).
• the definitive factor IX protein consists of 415 amino acids with 12 potential gamma-carboxyglutamic acid residues at amino acids 7, 8, 15, 17, 20, 21, 26, 27, 30, 33, 36 and 40. Two potential carbohydrate attachment sites occur at amino acid residues 157 and 167.
• the activation peptide encompasses residues 146-180, which are cut out in the activation of Factor IX (see Background of Invention) by the peptide cleavage of an R-A and R-V bond. This leaves a light chain spanning residues 1-145 and a heavy chain spanning residues 181-415.
• exons see Section H, above
• the exons are marked t, u, v, w, x, y, z. It can be seen that there is a rough agreement between the exon domains and the protein regions. For example, the exon for the signal peptide is distinct from that of the GLA region. Also that of the activation peptide is separated from the serine protease domain.
• the 3' non-coding region of the mRNA is extensive, consisting of 1390 residues (including the UAAUGA double terminator 1389-1394 but excluding the poly A tail).
• the factor IX cDNA is cleavable by the restriction enzyme Haelll to give a fragment from nuclotides 133-1440, i.e. a 1307 nuclotide long region of DNA entirely encompassing the definitive factor IX protein sequence.
• Haelll is GGCC.
• This fragment should be a suitable starting material for the expression of factor IX protein from suitable promoters in bacterial, yeast of mammalian cells.
• Another suitable fragment could be derived using the unique StuI site at residue 41 (corresponding to an early part of the hydrophobic signal peptide region) and linking it to a suitable promoter.
• the nucleotide sequence recognised by StuI is AGGCCT.
• Figure 13 shows the major bands obtained with DNA from normal individuals probed with either probe II ( Figure 6) or labelled 1.4 kbEcoRI fragment.
• Figure 6 With each of the 4 enzymes used, EcoRI, Hindlll, Bglll and Bell, a single major band of about 4.8, 5.2, 11 and 1.7 kb was obtained.
• the fact that these restriction fragments had the same length as those observed in the restriction map of clone lambda HIX-1 confirmed that the conditions of Southern blotting were precise enough to detect the factor IX gene in total DNA preparations. This provides the basis for analysis of DNA from the blood of patients with Christmas disease.
• a factor IX gene probe (this time pATIXcVII - the cDNA probe) was mixed with an irrelevant autosomal gene probe which was specific for the human A1 apolipoprotein (Shoulders and Baralle, Nucl.Acids Res. 10, 4873-4882, 1982).
• This experiment showed that patient 1 had the normal A1 apolipoprotein gene, characterised by a 12 kb band in an EcoRI digest, and confirmed that he lacked the 5.5 kb band observed with pATIXcVII and characteristic of the normal factor IX gene.
• E. coli K-12 strain 803 mentioned above which is a suitable host for the lambda HlX-lb phage, has been deposited at the NCIB on 26 July 1982 under Accession No. 11752. All deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• Wood Science & Technology (AREA)
• Molecular Biology (AREA)
• General Health & Medical Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Medicinal Chemistry (AREA)
• Microbiology (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Saccharide Compounds (AREA)
• Enzymes And Modification Thereof (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=26283513

Family Applications (1)

Country Status (4)

Cited By (9)

Families Citing this family (10)

• 1983
  • 1983-08-03 DE DE8383304487T patent/DE3380848D1/de not_active Expired
  • 1983-08-03 JP JP50256183A patent/JP2625412B2/ja not_active Expired - Lifetime
  • 1983-08-03 EP EP88116366A patent/EP0316558A1/en not_active Withdrawn
  • 1983-08-03 EP EP83304487A patent/EP0107278B1/en not_active Expired
  • 1983-08-03 WO PCT/GB1983/000191 patent/WO1984000560A1/en unknown
• 1996
  • 1996-10-23 JP JP8281135A patent/JP2764034B2/ja not_active Expired - Lifetime

Non-Patent Citations (5)

Also Published As

Similar Documents

Legal Events